Universal carrier for targeting molecules to Gb3 receptor expressing cells

ABSTRACT

The present invention concerns an universal polypeptidic carrier for targeting directly or indirectly a molecule to Gb3 receptor expressing cells and having the following formula STxB-Z(n)-Cys, wherein:
         STxB is the Shiga Toxin B subunit or a functional equivalent thereof,
           Z is an amino-acid devoided of sulfhydryl group, n being 0, 1 or a polypeptide,   Cys is the amino-acid Cysteine,
 
and the use thereof for MHC class I and MHC class II presentation of antigens.

This is a divisional of U.S. patent application Ser. No. 10/628,415filed Jul. 29, 2003, issued as U.S. Pat. No. 7,632,514 on Dec. 15, 2009,which is a continuation application of PCT Application No.PCT/EP02/01627 filed on Feb. 1, 2002, and which claims priority under 35U.S.C. §119(a) to Patent Application No. EP01400255.4 filed in Europe onFeb. 1, 2001, the entire contents of each of the above-identifiedapplications are hereby incorporated by reference.

The invention relates to a universal polypeptidic carrier for targetingmolecules to a Gb3 receptor for the B-subunit of Shiga-Toxin expressingcells and its use for intracellular transport and processing of saidmolecules.

Shiga Toxin is a bacterial toxin of the AB₅ subunit family that issecreted by Shigella dysenteriae. The A-subunit is the toxic moiety andinhibits the protein synthesis in higher eucaryotic target cells aftertransferring into the cytoplasm of said cells. The B-subunit is anhomopentamer protein (5B-fragments) and is responsible for toxin bindingto and internalization into target cells by interacting with theglycolipid Gb3 found on the plasma membranes of these cells. TheB-fragment is non toxic, but conserves the intracellular transportcharacteristics of the holotoxin which, in many Gb3 expressing cells, istransported in a retrograde fashion from the plasma membranes tocytosol, via endosomes.

The glycolipid Gb3 receptor has also been reported to be expressedpreferentially in some ectodermic derived tumors (plasma) and someBurkitt's lymphoma. It is also known as CD 77. In the present text, theterm Gb3 should be considered as an equivalent to CD77.

The authors have already shown that a CD8 human tumor Antigen fused tothe B subunit of Shiga toxin could efficiently be presented in an HLAclass I-restricted manner to specific CTL (1). This result wasindependently confirmed by another study that demonstrated that Shigaholotoxin, carrying a defined peptide epitope from influenza virus,could deliver the antigen into the MHC class I intracellular pathway(3).

The authors have also shown that fusion proteins between the Gb3receptor-binding non toxic B-fragment of bacterial Shiga toxin derivedfrom Shigella dysenteriae and an antigen, or an epitope from a modeltumor antigen, can elicit specific cytotoxic T lymphocytes response(CTL), whereas each moiety of said fusion protein does not leadindividually to CTL induction (1, 2, and WO 99/03881).

The difficulty of this technology is that, for each application, i.e.,for each antigen or fragment thereof, there is a need for a specificconstruction of a fusion protein, that necessitates a specificconstruction of a recombinant vector bearing the sequences encoding thisfusion protein to be expressed in a host cell.

The aim of the present invention is to overcome the above-mentioneddrawbacks and to provide a universal hook, or a universal carrier fortargeting a molecule to a Gb3 receptor expressing cell to enable thismolecule to be internalized, processed and/or expressed in said cellexpressing Gb3 receptor.

In the present invention, a Shiga toxin B-subunit (STxB) derivative, ormutant, termed STxB-Cys has been designed. In this protein, a Cysteineis added at the C-terminus of mature STxB. The protein, when purifiedfrom bacteria, carries the internal disulfide bond, as wild type STxB,while the sulfhydryl group at the C-terminal Cys is free. Due to theirnucleophilicity, free sulfhydryl groups are excellent acceptors fordirected coupling approaches (4).

Thus, the present invention relates to a universal polypeptidic carrierfor targeting directly or indirectly a molecule of interest to Gb3receptor expressing cells having the following formula: STxB-Z(n)-Cys,wherein:

STxB is the Shiga Toxin B subunit or a functional equivalent thereof,

Z is an amino-acid devoided of sulfhydryl group, n being 0, 1 or anamino-acid sequence,

Cys being the amino-acid Cysteine.

The STxB moiety of the universal carrier has the sequence described in(8) or a functional equivalent thereof. A functional equivalent means apolypeptidic sequence having the capacity to bind specifically to theGb3 receptor and/or to trigger an internalization of an antigen and itspresentation in an MHC class-I restricted pathway, or both MHC class Iand class II on the same antigen presenting cell.

In the light of the heterogeneity of expression of tumor antigens, theallele-specific loss of MHC class I expression at the surface of tumorcells, and the necessity to have concomitant presentation of antigens byboth MHC class I and class II on the same antigen presenting cell, it isadvantageous to couple full size antigen proteins to the B-subunit fortargeting to dendritic cells.

In a preferred embodiment, n is 0 and the universal carrier has thefollowing sequence (SEQ ID No 1):

COOH-MKKTLLIAASLSFFSASALATPDCVTGKVE YTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFRC-NH2

As a matter of fact, if the Z linker is too long, i.e., when n is equalor greater than 2, some internal disulfide bridges might occur, andprevent either the binding of STxB to the Gb3 receptor and especiallyprevent the binding to the molecule of interest.

According to the invention, the molecule of interest is selected in thegroup constituted of proteins, peptides, oligopeptides, glycoproteins,glycopeptides, nucleic acids, polynucleotides, or a combination thereof.

In another aspect of the invention, the molecule of interest is anantigen to be targeted to antigen presenting cells. Such cells areselected in a group comprising T lymphocytes, dendritic cells,macrophages Langerhans cells and the like.

In another aspect of the invention, the molecule of interest are drugssuch as haptenes, psoralenes, or any compounds provided that they have achemical group linkable with the —SH group of the Cysteine moiety ofSTxB-Cys.

The drug might be linked either directly or after activation withcompounds such as bromoacetate, or any other method known by a skilledperson, provided that the result of the reaction is a chemical entityhaving the following formula: STxB-Cys-M, M being all the abovementioned molecules of interest.

The coupling approaches for covalent binding of a peptidic or apolypeptidic moiety to STxB-Z(n)-Cys can be any method or processesdescribed or carried out by a skilled person.

A first method that can be embodied is the use of SPDPhetero-bi-functional cross-linker described par Carlsson et al (5).However, SPDP is capable of being cleavable by serum thiolases that is acause of decreasing the yield of the reaction.

A second method for covalent coupling of STxB-Z(n)-Cys peptides withanother peptide of interest is to produce bromoacetyl or maleimidefunctions on the latter as described by P. Schelte et al (4). Briefly,the peptide of interest is chemically activated with bromoacetateanhydride or by a maleimide group respectively. In appropriate reactionconditions (pH, temperature, incubation times), these groups areeliminated by cis-elimination, yielding respectively to —S—S, —S—CH₂—,to —S—CO— or to —S—NH— covalents linkages.

As an example, the polypeptide or the peptide to be coupled to the —SHmoiety the C-terminal Cysteine of the universal carrier, has itsN-terminus activated with bromoacetic anhydride following the reactionscheme:

Br—CH₂—CO—O—CO—CH₂—Br+NH₂-peptide

Br—CH₂—CO—NH-peptide+Br—CH₂—COOH

The Bromoacetyl function has high chemoselectivity for peptide thiolgroups and the activated peptide can be reacted with STxB-Cys asfollows:

STxB-Cys-SH+Br—CH₂—CO—NH-peptide

STxB-Cys-S—CH₂—CO—NH-peptide+HBr

The resulting thioether-linkage is stable to hydrolysis.

Another method for coupling a molecule to the universal carrier of theinvention is to use MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester)as shown in FIG. 6 and explained in example 5. This coupling allows thetransport and processing of large molecules such antigenic proteins orglycoproteins through MHC class I and/or MHC class II pathways.

Thus, another aspect of the invention is the product resulting from acovalent binding of STxB-Z(n)-Cys with a molecule of interest by a—S—S—, —S—CO—, or S—CH2- or —S—NH— linkage.

In one embodiment, the molecule of interest to be targeted to an antigenpresenting cells is constituted by or comprises a polypeptidicstructure, such an antigens or epitopes thereof, glycopeptides orglycoproteins, lipopeptides or lipoproteins.

In a preferred embodiment, the product resulting from the coupling ofSTxB-Z(n)-Cys with an antigen or a fragment thereof, where (n) is 0, 1,or 2, and preferably 0, is able to be presented in an MHC class I andMHC class II restricted pathway.

In another embodiment, the molecule of interest is a polypeptide capableof binding with polynucleotide structures such as DNA or RNA molecules.Such molecules might be vectors or plasmids comprising a sequence ofinterest to be expressed in a target cell. In the present invention, atarget cell is a eucaryotic cell bearing on its membrane the Gb3receptor.

Thus, the universal carrier of the present invention is also a carrierfor introducing a nucleotide sequence in a target cell either for genetherapy or for obtaining recombinant cells expressing heterologousproteins.

In another embodiment, the universal carrier according to the presentinvention can be operably linked directly through a covalent binding orindirectly through a linker to a cytotoxic drug to be targeted to tumorcells expressing Gb3 receptor.

The term “indirect binding” means that the universal carrier iscovalently linked through the sulfhydryl moiety of the C-terminalCysteine to a linker, said linker being operably linked to a drug or apro-drug to be internalized into Gb3 receptor bearing cells.

This linkage might be a covalent binding or a non-covalent binding,provided that the affinity between the linker and the drug (or thepro-drug) is higher than 10⁻⁹ mole/l.

Another aspect of the invention is an isolated polynucleotide selectedfrom the group of:

(a) a polynucleotide comprising the nucleotide sequence STxB encodingthe Shiga Toxin B subunit or a functional equivalent thereof bearing atits 3′ end the codon TGT, or the codon TGC encoding Cysteine;

b) a polynucleotide comprising a nucleotide sequence having at least 80%sequence identity to a nucleotide sequence encoding the Shiga Toxin Bsubunit or a functional equivalent thereof bearing at its 3′ end thecodon TGT or TGC; and

c) a nucleotide sequence complementary to the sequence in a) or b).

In a preferred embodiment, the polynucleotide has the following SEQ IDNO 2:

5′-atgaaaaaaacattattaatagctgcatcgctttcatttttttcagcaagtgcgctggcgacgcctgattgtgtaactggaaaggtggagtatacaaaatataatgatgacgatacctttacagttaaagtgggtgataaagaattatttaccaacagatggaatcttcagtctcttcttctcagtgcgcaaattacggggatgactgtaaccattaaaactaatgcctgtcataatggagggggattcagcgaagttatttttcgttgt-3′

The present invention relates also to a recombinant vector or to aplasmid comprising a polynucleotide sequence as described above, andcapable of expressing the universal carrier STxB-Z(n)-Cys, where (n) is0, 1 or 2, STxB and Z have the same significance as above, in anappropriate host cell.

As an example, a convenient vector is the plasmid pSu108 described in(7).

Another object of the present invention is to provide a method forobtaining a plasmid expressing STxB-Z(n)-Cys comprising:

a) providing a plasmid comprising a STxB sequence;

b) applying two PCR amplification steps using two couples of primers,AA′ and BB′,

-   -   A and B being complementary to each other and comprising the Cys        codon,    -   A′ and B′ being outside the STxB sequence;

c) isolating the amplified fragments;

d) hybridizing the amplified fragments;

e) applying a PCR amplification on the hybridized fragments;

f) insertion of the amplified fragment into a plasmid.

In a preferred embodiment, the plasmid pSU108 (7) containing STxBfragment was modified to introduce the Cysteine codon TGT at the 3′ endof the B-fragment cDNA. The primers for step b) are respectively for AA′and BB′:

primer A: (SEQ ID 3) 5′-AGCGAAGTTATTTTTCGTTGTTGACTCAGAATAGCTC-3′, andprimer B: (SEQ ID n^(o) 4) 5′-GAGCTATTCTGAGTCAACACGAAAAATAACTTC-3′.primer A′: primer ShigaAtpE: (SEQ ID n^(o) 5) 5′-CACTACTACGTTTTAAC-3′,primer B′: primer Shiga-fd: (SEQ ID n^(o) 6) 5′-CGGCGCAACTATCGG-3′.

The PCR of step e) yields a fragment that is cloned into the SphI andSalI restriction sites of pSU108. Sequences derived by PCR are verifiedby dideoxy-sequencing.

The skilled person can easily design the choice of primers, plasmids forproducing a vector bearing the polynucleotide sequence expressingSTxB-Z(n)-Cys in an appropriate host cell, provided that this successionof steps allows the interpretation of the Cys codon into the amplifiedfragment.

The invention also provides a recombinant cell line obtained bytransformation with the recombinant vector containing the polypeptidesequence encoding the universal carrier as described above. In apreferred embodiment, said recombinant cell line is a procaryotic cell,preferentially E. coli.

In a still preferred embodiment, the plasmid is pSU108 having SEQ ID No.2 integrated between the SphI and SalI restriction sites, and thecorresponding cell line has been deposited at CNCM on Dec. 19, 2000 withthe registration number I-2604.

The present invention also provides a process for producing a universalcarrier as described above comprising:

a) culturing a recombinant cell line as described above,

b) obtaining a periplasmic extract of said cells, and

c) purifying said polypeptide.

Preferentially, the cell line is E. coli and in c) the purification ismade by anion exchange column chromatography followed by a gelfiltration column chromatography.

Such a process is particularly advantageous for large scale productionof the universal carrier, as far as it can then be operably linked bycovalent coupling with a molecule of interest, and used within a largescope of application.

The present invention also provides a method for delivering a sequenceof interest into the MHC class I pathway using a product obtained bycovalent binding of the Cys moiety of the universal carrier with saidsequence of interest; this method is advantageous to elicit a CTLresponse to a given antigen or epitope thereof as far as the product isspecific to the cell involved in the MHC class I pathway.

As a matter of fact, the inventors have shown that an immunodominantpeptide, derived from the ovalbumin protein, and coupled chemically toSTxB-Cys, could be presented by antigen presenting cells to specifichybridoma cells, demonstrating that STxB could deliver exogenousimmunogenic peptide in the MHC class I pathway. To exclude a bias due tothe presence of free peptides contaminating the material, experimentsusing fixed dendritic cells clearly demonstrated that theinternalization of the fusion protein was required for this process. Theinventors also have shown that the Shiga toxin receptor, Gb₃, was alsoinvolved in the ability of STxB-Cys to target exogenous peptide in theendogenous MHC class I pathway.

The invention also pertains to a method for delivering an expressionvector containing a sequence of interest into a Gb3 receptor expressingcells characterized in that said expression vector is bound to a lysinerich peptide covalently linked to the Cys moiety of the universalcarrier.

As an example, the lysine rich peptide is a 16-mer poly-lysine which isable to bind any polynucleotidic sequence, either of DNA or RNA nature.Such a peptide carrying a 16-mer of lysines will be activated at itsN-terminus by bromoacetate anhydride and coupled to STxB-Cys. Expressionplasmids will be bound to this coupling product, and vectorization ofDNA into target cells is assayed using convenient reporter systems, suchas the green fluorescent protein or luciferase.

The capacity to target expression plasmids with the help of STxB to thenucleus of antigen presenting cells is expected to further improve thepower of this vector, since i) DNA can even more easily be adopted tonew experimental or clinical needs, and ii) due to its potentiationeffect, expression of antigenic peptides or proteins from DNA wouldfurther increase the sensitivity of STxB-dependent antigen presentation.

The invention also provides a method for delivering a drug or a pro-druginto a cell, particularly into a cancer cell bearing Gb3 (or CD77)receptor.

The glycolipid Gb₃ receptor has been reported to be preferentiallyexpressed in some neuroectodermic derived tumors (glioma) and someBurkitt's lymphoma. Since one limitation of the use of chemotherapy incancer is secondary side effects of the drugs because of their toxicityon normal cells, the drugs are preferentially vectorized in tumor cellsby using STxB-Cys. The drugs are activated to become reactive with thesulfhydryl group of STxB-Cys. To achieve this, a maleimide group can beintroduced on a drug, for example psoralenes compounds.

The present invention also pertains to:

a pharmaceutical composition for enhancing the immunogenicity of apeptide or a protein or a glycoprotein or a lipopeptide, containing theuniversal carrier covalently linked by its Cys moiety to said peptide orprotein or glycoprotein or lipopeptide;

a pharmaceutical composition for treating tumor cells bearing the Gb3receptor (CD77), containing the universal carrier according to theinvention covalently linked by its Cys moiety to a drug or a pro-drugtoxic for said tumor cells.

Without limiting the scope of the universal carrier of the invention andits widespread use in different applications, the hereinafter examplesand figures illustrate the advantages of the present invention.

LEGEND OF THE FIGURES

FIG. 1 represents the protein profile of the final SephaDex 75 columnyielding purified STxB-Cys. Fractions 20-25 contain mostly monomericSTxB-Cys (the positions of monomeric and dimeric STxB-Cys are indicatedto the right). Molecular weight markers are indicated to the left.

FIG. 2 a represents the coupling of Type2 of Pep2 [as defined in example2] to STxB-Cys, followed by an in vitro antigen presentation assay on D1dendritic cells, as described in (2). Two different preparations ofSTxB-Cys coupled to the SL8 peptide, an immunodominant epitope ofovalbumin were used (termed 4A and 9A). Upon fixation, antigenpresentation is abolished showing that no extracellular processingoccurred.

FIG. 2 b represents a control experiment of FIG. 2 a, in which it isshown that fixed D1 can still present free SL8 peptide.

FIG. 3 represents another experiment on fixed and non fixed D1 cellsusing a coupling reaction of Type1 on STxB-SH and Pep1 [as defined inexample 2].

FIG. 4 represents the B-subunit dependent presentation of antigenicpeptides derived from a coupling of Pep2 to B-Glyc-Cys-KDEL. The use ofFab fragments from an antibody that neutralize STxB binding to Gb3 alsoabolishes antigen presentation.

FIG. 5 a shows that the Gb3 synthesis inhibitor PPMP inhibits B-subunitdependent antigen presentation and FIG. 5 b shows that SL8 presentationis not decreased in PPMP treated cells.

FIG. 6 represents reaction scheme for full size Ova coupling to STxB-Cysusing the heterobifunctional cross linker MBS. Top: first reactionlinking MBS to primary amines of Ova. Middle: second reaction betweenactivated Ova and STxB-Cys. Bottom: Structure of MBS.

FIG. 7 shows western analysis of Ova coupling to STxB-Cys. The upperpart of the figure represents an immunoblot using anti-STxB antibody,the lower part an immunoblot using anti-Ova antibody. The intermediatesof different steps of the purification procedure are shown. Lane 1:uncoupled proteins (marked by a cross). Lane 2: coupling reaction(coupling product marked by an arrow). Lane 3: eluate of theimmunoaffinity column doted with anti-STxB antibody. Lanes 4-7:fractions from the gelfiltration column. Lane 4: fractions 9-10. Lane 5:fractions 11-12. Lane 6: fractions 13-14. Lane 7: fractions 15-19 (freeSTxB-Cys). Fractions 11-12 contain the bulk of monomeric couplingproduct. Some material with lower electrophoretic mobility can also bedetected, originating likely from dimeric Ova present in the originalpreparation.

FIG. 8 represents immunofluorescence analysis of STxB-Cys-Ova transportin HeLa cells. The coupling product STxB-Cys-Ova (upper part of thefigure) or a mixture of STxB-Cys and Ova (lower part of the figure) wereincubated with HeLa cells on ice. After washing, the cells were shiftedfor 45 min to 37° C., fixed, and stained with the indicated antibodies.Note that when Ova is linked to STxB-Cys (top), the protein isvectorized into the Golgi apparatus, co-stained for the Golgi markerRab6. If Ova is only mixed with STxB-Cys (bottom), the protein cannot bedetected on the cells.

FIG. 9 a shows MHC class I and II restricted antigen presentation usingSTxB-Cys-Ova₃₂₉₋₃₃₉ used to sensitize the murine dendritic cell line D1.

FIG. 9 b shows MHC class I and II restricted antigen presentation usingSTxB-Cys-Ova₂₅₇₋₂₆₄ and the immunodominant SL8 Ova-derived peptide aloneused to sensitize the murine dendritic cell line D1.

EXAMPLE 1 Preparation of the Universal Carrier

a) Construction of a Plasmid Expressing STxB-Cys:

In a preferred embodiment, the plasmid pSU108 (7) was modified tointroduce the Cysteine codon tgt at the 3′ end of the B-fragment cDNA.PCR primer A: SEQ ID NO. 3 (5′-AGCGAAGTTATTTTTCGTTGTTGACTCAGAATAGCTC-3′)and primer A′: SEQ ID NO. 4 (5′-GAGCTATTCTGAGTCAACACGAAAAATAACTTC-3′)were used with plasmid specific primers ShigaAtpE: SEQ ID NO. 5(5′-CACTACTACGTTTTAAC-3′) and Shiga-fd: SEQ ID NO. 6(5′-CGGCGCAACTATCGG-3′) to produce DNA fragments which, in a second PCRwith primers Shiga AtpE and Shiga-fd yielded a fragment that was clonedinto the SphI and SalI restriction sites of pSU108. Sequences derived byPCR were verified by dideoxy-sequencing.

b) Protein Purification:

b) 1. Preparation of the periplasmic extract was performed as follows:

-   -   Inoculate 125 ml of LB/Amp with 125 μl of an overnight culture        grown at 30° C.,    -   grow over night at 30° C.,    -   transfer into 375 ml of LB/Amp at 50° C.; incubate 4 hours at        42° C.,    -   centrifuge to pellet cells,    -   wash cells 3 times with 10 mM Tris/HC1, pH 8.0,    -   re-suspend cells in 200 ml of 25% sucrose, 1 mM EDTA, 10 mM        Tris/HC1, Ph 8.0; incubate at room temperature for 10 min.,    -   centrifuge to pellet cells,    -   re-suspend cells in 200 ml of ice cold water containing a        protease inhibitor cocktail; incubate on ice for 10 min.,    -   centrifuge; collect supernatant; add 20 mM Tris/HC1, Ph8.0.

b) 2. Purification on columns:

The periplasmic extract was loaded on a QFF anion exchanger column(pharmacia) and eluted at 230 mM Nacl. STxB-Cys containing fractions arepooled, diluted 4-fold and loaded on a Mono Q anion exchanger column(pharmacia), followed by elution at 230 mM Nacl. After concentrationwith microconcentration devices from PallFiltron, the pooled fractionswere passed through a Sephadex 75 gel filtration column. Purity wasabove 95% (FIG. 1).

b) 3. Product characterization:

The B-fragments of STxB-Cys, purified from Sephadex 75 gel filtrationcolumns, are essentially monomeric (FIG. 1). This is in markeddifference to constructions where the Cysteine was added at more than 2amino acids from the natural C-terminus of the B-fragment. In thesecases, neighbouring B-fragments within a pentamer are engaged indisulfide bonds.

EXAMPLE 2 Conditions for Coupling of Activated Peptides to the UniversalCarrier

a) Carriers:

Three different carriers have been compared.

1) STxB-Cys: B-fragment to which a Cys has been added right to itsC-terminus. This protein elutes as a monomer from the purificationcolumns.

2) STxB-Z₂-Cys: carrier with a short spacer (2 amino acids resultingfrom a cloning cassette) between the C-terminus of the wild typeB-fragment and the Cys. The majority of the protein eluted as dimersfrom the purification columns. These can be separated under reducingconditions, indicating the formation of disulfide bonds between monomersin the pentameric B-subunit complex.

3) STxB-Glyc-Cys-KDEL: carrier in which the Cys is located between aGlycosylation cassette being 9 amino acid long and a C-terminal KDELpeptide. The majority of the protein eluted as dimers from thepurification columns. These can be separated under reducing conditions,indicating the formation of disulfide bonds between monomers in thepentameric B-subunit complex.

b) Test Peptides:

1) Pep1: a synthetic peptide of 16 amino acids carrying the SL8antigenic peptide derived from chicken ovalbumin.

2) Pep2: a synthetic peptide of 24 amino acids as above with, inaddition, a His-gag at its C-terminus.

3) SL8: the antigenic peptide from ovalbumin that can directly exchangewith peptides on MHC class I complexes at the plasma membrane of antigenpresenting cells.

c) Coupling Conditions:

Under reducing conditions (Type1): Fusion proteins were treated with DTTovernight, then activated peptide (carrying a bromo acetate group at itsN-terminus) was added in excess. Conditions used for the first couplingexperiments using fusion proteins will mostly dimerize monomers(proteins STxB-Z₂-Cys and STxB-Glyc-Cys-KDEL).

Under non-reducing conditions (Type2): Fusion proteins are directlyreacted with the activated peptides.

d) Biochemical and Morphological Controls:

Pep2 carries a His-tag. This has allowed us, using an anti-His antibody,to show the presence of Pep2 on B-subunit by Western blotting, and theB-subunit dependent transport of Pep2 in HeLa cells.

FIG. 2 a shows that a dose dependent stimulation of the B3Z CTLhybridoma (measurement of β-galactosidase activity) was observed withnon-fixed cells, while fixation abolished antigen presentation.

Note that antigen presentation only works on non-fixed cells, indicatingthat the observed presentation does not result from contaminating freePep2.

FIG. 2 b shows the control experiment of FIG. 2 a in which it is shownthat fixed D1 cells can still present free SL8 peptide.

In FIG. 3, it appears that in this type of protocol, some free Pep1appears to co-purify with the fusion protein, since at high doses(200-1000 nM), some presentation was observed on fixed cells.Presentation by SL8 is shown to the right.

In FIG. 4, the coupled protein (lanes 1 and 2) or the SL8 Peptide (lanes5 and 6) were incubated (lanes 2 and 5) or not (lanes 1 and 4) withanti-B-subunit Fab-fragment derived from the 13C4 antibody whichinhibits the binding of the B-subunit to Gb3. Note that the Fab-fragmentneutralizes the capacity of the B-subunit to introduce the antigenicpeptide into the class I pathway, while the presentation with SL8 is notaffected under these conditions. The background signal in thisexperiment was at 0.3.

In FIG. 5 a, D₁ cells were pre-treated with PPMP (see FIG. 3 b of (2))for 3 days. This treatment lead to an important decrease of Gb3expression at the cell surface, without however eliminating itcompletely. Under this condition, antigen presentation from a couplingreaction of Pep1 with STxB-Glyc-Cys-KDEL was significantly reduced,indicating that Gb3 is important for the presentation phenomena.

It appears from all these experiments that the coupling undernon-reducing to STxB-Cys is surprisingly efficient (in terms ofsensitivity; note that, as shown in FIG. 1, only 4 nM of STxB-Cys-Pep2are necessary to have a response). Thus, the universal carrier STxB-Cysis preferred due to its simplicity in its preparation and to thereproducibility of the coupling.

Hence, the optimal conditions for coupling of activated peptides toSTxB-Cys were the following:

dialyse STxB-Cys against 20 mM Borate buffer, pH 9.0, 150 mM NaCl,

concentrate to 1 mg/ml,

dissolve N-terminally activated peptide (activated with bromoacetateanhydride) at 12 mM in DMSO,

dilute peptide to 0.2 mM in protein solution,

incubate 12 hours at room temperature,

dialyse against PBS.

EXAMPLE 3 Characterization of STxB as to its Antigen PresentationCapacity

The following experimental series will help to fully describe thecapacity of STxB to function in antigen presentation system.

a) Class I- and Class II-Restricted Antigen Presentation:

A peptide carrying class I- and II-restricted antigenic peptides fromchicken ovalbumin Br—CH2-CO—NH-LEQLESIINFEKLTEWSLKISQAVHAAHAEINEAGR (SEQID NO: 6) sequences 257-264 and 323-339 were coupled to STxB-Cys, andthe class I- and class II-restricted presentation of these peptides wereassayed using the corresponding T-cell hybridomas.

b) Coupling of Whole Size Proteins.

Our preliminary evidence suggests that chicken ovalbumin can be coupledto STxB-Cys. These experiments have been done with the SPDPheterobifunctional cross-linker. (Carlsson et al., 1978).

A first series of antigen presentation experiments indicated that theovalbumin protein can be introduced into the endogenous MHC classI-restricted antigen presentation pathway of mouse dendritic cells. SPDPhas the inconvenience of being cleavable by serum thiolases. Thiscross-linker was successfully substituted by MBS which is non-cleavable.Other antigenic proteins (Mart 1 and polypeptides derived from HPV16-E7and Muc1) are tested to show that the procedure is of universal use.

c) Coupling of Complex Protein Mixtures.

A lysate from the cervix carcinoma-derived cell line Caski is used. Thiscervix carcinoma cell line, which expresses the HLA-A2 allele at itsmembrane, also expresses Human papillomavirus derived peptides. E7 is aearly transcribed ORF from HPV which is necessary for transformation ofprimary keratinocytes. Since anti-E7 HLA A2-restricted CTL are elicitedin vitro. The efficacy of the coupling of this protein mixture by apresentation assay specific for HLA-A2 E7 derived peptides was tested.As control, a lysate from a HLA-A2-positive cell line which does notexpress E7 (croft cells or Daudi) was coupled to STxB-lys.

EXAMPLE 4 Application to MHC Class I-Restricted Antigen Presentation

The experiment of FIG. 4 shows that STxB-Cys dependent antigenpresentation is inhibited when the interaction with Gb₃ is abolished.Here it is found that pre-binding of the Fab fragment of monoclonal Abagainst STxB to 0.1 μM STxB-Cys, coupled to SL8, inhibited antigenpresentation, suggesting that STxB-Cys binding to Gb₃ is necessary forantigen presentation. Similar results were obtained when Gb₃-expressionwas inhibited with a drug (FIG. 4).

EXAMPLE 5 Reaction Chain for Coupling Ovalbumine to the STxB-Cys

The reaction scheme is shown in FIG. 6.

In a first reaction, the N-hydroxysuccinimide ester moiety of MBS reactswith primary amines on an antigenic target protein, such as the modelprotein ovalbumin (Ova). The reaction product is purified and thenincubated in a second reaction with STxB-Cys leading to coupling via themaleimidobenzoyl moiety.

FIG. 7 shows the SDS-PAGE and Western analysis of a typical couplingreaction involving STxB-Cys and Ova. For coupling, 20 mg/ml of Ova in100 mM HEPES, pH 7.4, was incubated with 4.5 mM of MBS for 30 min atroom temperature. The reaction is passed through a PBS/EDTA 10 mMequilibrated gel filtration column. Eluted Ova is concentrated to 20mg/ml. 1 volume of STxB-Cys at 3.5 mg/ml in PBS/EDTA is mixed with 1volume of activated Ova and incubated over night at room temperature.

FIG. 7 shows that within the coupling reaction, bands with lowerelectrophoretic mobility (labeled with arrows; coupling product) can bedetected in addition to uncoupled STxB (upper part) and uncoupled Ova(lower part; uncoupled proteins are labeled with a cross). The reactionproduct is purified by passage through an immunoaffinity column madewith 13C4 anti-STxB monoclonal antibody (lane IP column). Note that freeOva is eliminated. Eluted STxB-Cys (coupled and non-coupled) is thenpassed through a gel filtration column to separate free STxB (fractions15-19) from coupled STxB-Cys (fractions 9-14; note that fractions 11-12contain the bulk of coupled protein; the upper coupling band, which isminor compared to the lower band, probably results from dimeric Ova).

EXAMPLE 6 Intracellular Transport Characteristics of STxB-Cys-OvaCoupling Product

0.5 μM of STxB-Cys-Ova was incubated with HeLa cells on ice. The cellswere washed and shifted to 37° C. for 45 min, fixed, and stained for theindicated antibodies. As shown in FIG. 8, when STxB-Cys and Ova werelinked by MBS, Ova immunoreactivity could be detected together with STxBimmunoreactivity in the Golgi apparatus, stained by Rab6. When bothproteins are incubated as separate entities with HeLa cells, onlySTxB-Cys is transported to the Golgi, while Ova cannot be detected onthe cells. These data clearly show that couples STxB-Cys is stilltransported in the same manner as uncoupled STxB-Cys, and that Ova isvectorized via STxB-Cys.

EXAMPLE 7 The STxB-Cys Allows Both, MHC Class I and II RestrictedPresentation of Peptides Derived from Full Size Exogenous AntigenicProteins

In a first experiment (FIG. 9, left), we have shown that whenSTxB-Cys-Ova is used to sensitize the murine dendritic cell line D1, aclear increase of the presentation of the MHC class II restricted Ovaderived peptide Ova₃₂₃₋₃₃₉ is observed, compared to D1 cells pulsed withnon-vectorized Ova. Indeed, a significant presentation of Ova₃₂₃₋₃₃₉peptide could be detected with as little as 0.01 nM of STxB-Cys-Ova,whereas 10 nM of full size Ova was required for an efficientpresentation of the same peptide. The presentation of the IA^(b)restricted Ova₃₂₃₋₃₃₉ peptide was revealed using the B097.10 hybridomathat produces IL-2 after recognition of this peptide. As a control, wedid not observe IL-2 secretion when an irrelevant MHC class IIrestricted hybridoma was used instead of B097.10 (data not shown).

In a second experiment, we have pulsed the same D1 dendritic H2^(b)restricted cell line with either Ova alone or with STxB-Cys-Ova. Nopresentation of the Ova-derived immunodominant SL8 peptide (Ova₂₅₇₋₂₆₄)was observed when the D1 cells were sensitized with up to 100 nM of freeOva, while 1-10 nM of STxB-Cys-Ova allowed the presentation of the SL8peptide, as revealed by the specific B3Z hybridoma that recognize theSL8 peptide in the context of K^(b) molecules. As a control, it wasshown that no activation of an irrelevant hybridoma was observed underthe same experimental conditions.

Altogether, these results clearly demonstrate that STxB-Cys targets fullsize proteins with high efficiency into both, the MHC class I and classII pathways.

BIBLIOGRAPHY

-   (1) Ren-Shiang Lee, Eric Tartour, Pierre van der Bruggen, Valérie    Vantomme, Isabelle Joyeux, Bruno Goud, Wolf Herman Fridman and    Ludger Johannes, “Major histocompatibility complex class I    Presentation of exogenous soluble tumor antigen fused to the    B-fragment of Shiga toxin”. Eur. J. Immunol. (1998) 28: 2726-2737.-   (2) Nacilla Haicheur, Emmanuelle Bismuth, Sophie Bosset, Olivier    Adotevi, Guy Warnier, Valérie Lacabanne, Armelle Regnault, Catherine    Desaymard, Sebastian Amigorena, Paola Ricciardi-Castagnoli, Bruno    Goud, Wolf H. Fridman, Ludger Johannes and Eric Tartour, “The B    Subunit of Shiga Toxin Fused to a Tumor Antigen Elicits CTL and    Targets Dendritic Cells to Allow MHC Class I-Restricted Presentation    of Peptides Derived from Exogenous Antigens”. The Journal of    Immunology (2000) 165: 3301-3308.-   (3) Noakes, K. L., H. T. Teisseranc, J. M. Lord, P. R. Dunbar, V.    Cerundolo and L. M. Roberts “Exploiting retrograde transport of    Shiga-like toxin 1 for the delivery of exogenous antigens into the    MHC class I presentation pathway”. Febs. Lett. (1999) 453:95.-   (4) Philippe Schelté, Christophe Boeckler, Benoît Frisch and Francis    Schuber “Differential Reactivity of Maleimide and Bromoacetyl    functions with Thiols: Application to the Preparation of Liposomal    Diepitope Constructs”. Eur. J. Immunol. (1999) 29:2297-2308.-   (5) Carlsson, J., H. Drevin, and R. Axen. 1978. Protein thiolation    and reversible protein-protein conjugation. N-Succinimidyl    3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.    Biochem. J. 173:723-737.-   (6) Su, G. F., H. N. Brahmbhatt, J. Wehland, M. Rohde and K. N.    Timmis “Construction of stable LamB-Shiga toxin B subunit hybrids:    analysis of expression in Salmonella typhimurium aroA strains and    stimulation of B subunit-specific mucosal and serum antibody    responses”. (1992) Infect. Immun. 60:3345-3359.-   (7) Johannes, L., Tenza, D., Antony, C. and Goud, B., “Retrograde    transport of KDEL-bearing B-fragment of Shiga toxin”. (1997) J.    Biol. Chem. 272: 19554-19561.-   (8) N. A. Stockbine, M. P. Jackson, L. M. Sung, R. K. Holmes, A. D.    O'Brien, J Bacteriol 170, 1116-22 (1988).

The invention claimed is:
 1. A method for delivering a sequence ofinterest into the MHC class I and MHC class II pathways said methodcomprising: administering a universal polypeptidic carrier and havingthe following formula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin Bsubunit or a functional equivalent thereof, Z is an amino acid devoid ofa sulfhydryl group, n being, 0, 1 or a polypeptide, Cys is the aminoacid cysteine, wherein said sequence of interest is bound to saiduniversal polypeptidic carrier via the Cys moiety, wherein saiduniversal polypeptide binds to Gb3 receptor expressing cells, whilemaintaining universal polypeptide conformation and enters into the MHCclass I and MHC class II pathways.
 2. The method according to claim 1,wherein said sequence of interest is covalently bound to said Cys moietyon said universal polypeptidic carrier.
 3. The method according to claim1, wherein said sequence of interest is selected from the group of: asequence encoding an immunogenic peptide, a sequence encoding acytotoxic drug, a prodrug and a sequence encoding a therapeuticallyactive molecule.
 4. The method according to claim 1, wherein thesequence of interest is an antigen to be targeted to antigen presentingcells.
 5. A method for delivering an expression vector containing asequence of interest into Gb3 receptor expressing cells, said methodcomprising: administering a universal polypeptidic carrier and havingthe following formula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin Bsubunit or a functional equivalent thereof, Z is an amino acid devoid ofa sulfhydryl group, n being 0, 1 or a polypeptide, Cys is the amino acidcysteine, wherein said expression vector is operably linked to alysine-rich peptide which is covalently hound to said universalpolypeptidic carrier via the Cys moiety, wherein said universalpolypeptide binds to Gb3 receptor expressing cells, while maintaininguniversal polypeptide conformation and enters into the MHC class I andMHC class II pathways.
 6. The method according to claim 5, wherein saidsequence of interest is selected from the group of: a sequence encodingan immunogenic peptide, a sequence encoding a cytotoxic drug, a prodrugand a sequence encoding a therapeutically active molecule.
 7. The methodaccording to claim 5, wherein the sequence of interest is an antigen tobe targeted to antigen presenting cells.
 8. A method for delivering asequence of interest into the MHC class I and MHC class II pathways saidmethod comprising: administering a universal polypeptidic carrier andhaving the following formula STxB-Z(n)Cys, wherein: STxB is the ShigaToxin B subunit or a functional equivalent thereof, Z is an amino aciddevoid of a sulfhydryl group, n being 0, 1 or a polypeptide, Cys is theamino acid cysteine, wherein said sequence of interest is bound to saiduniversal polypeptidic carrier via the Cys moiety and wherein themolecule of interest is covalently bound to said Cys moiety on saidpolypeptidic carrier through a —S—S—, —S—CO, or S—NH linkage.
 9. Amethod for delivering an expression vector into the MHC class I and MHCclass II pathways said method comprising: administering a universalpolypeptidic carrier and having the following formula STxB-Z(n)-Cys,wherein: STxB is the Shiga Toxin B subunit or a functional equivalentthereof, Z is an amino acid devoid of a sulfhydryl group, n being 0, 1or a polypeptide, Cys is the amino acid cysteine, wherein saidexpression vector is bound to said universal polypeptidic carrier viathe Cys moiety and wherein the molecule of interest is covalently boundto said Cys moiety on said polypeptidic carrier through a —S—S—, —S—CO,S—CH2 or S—NH linkage.
 10. A method for delivering a sequence ofinterest into the MHC class I and MHC class Ii pathways said methodcomprising: administering a universal polypeptidic carrier and havingthe following formula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin Bsubunit or a functional equivalent thereof, Z is an amino acid devoid ofa sulfhydryl group, n being 0, 1 or a polypeptide, Cys is the amino acidcysteine, wherein said sequence of interest is bound to said universalpolypeptidic carrier via the Cys moiety and wherein the molecule ofinterest is an antigen to be targeted to T lymphocytes, dendritic cells,macrophages or Langerhan cells.
 11. A method for delivering anexpression vector into the MHC class I and MHC class II pathways saidmethod comprising: administering a universal polypeptidic carrier andhaving the following formula STxB-Z(n)-Cys, wherein: STxB is the ShigaToxin B subunit or a functional equivalent thereof, Z is an amino aciddevoid of a sulfhydryl group, n being 0, 1 or a polypeptide, Cys is theamino acid cysteine, wherein said expression vector is bound to saiduniversal polypeptidic carrier via the Cys moiety and wherein themolecule of interest is an antigen to be targeted to T lymphocytes,dendritic cells, macrophages or Langerhan cells.